PROJECT SUMMARY Despite some remarkable advances in treating certain types of cancer, the nature of mutations and heterogeneity of acute myeloid leukemia (AML) have made this cancer challenging to treat successfully. Genomic data have suggested many new therapeutic targets, but even successful molecular inhibition does not always translate to effective therapy, justifying a broader understanding of mechanisms and pathways altered in AML. Our recent studies illuminated a novel pathway by which the endogenous MLL1 and MLL2 histone methyltransferases contribute to leukemogenesis driven by MLL fusion oncoproteins. In MLL-AF9 or MLL-AF6-driven AML, we found that the endogenous MLL2 histone methyltranferase played a major role in sustaining several coordinated pathways that enhance leukemia survival and proliferation. Although MLL1 did not contribute on its own to leukemogenesis, it collaborated with MLL2 in regulating critical AML survival/proliferation pathways affecting NF?B, integrin ?3 and IL-3 signaling. Given the widespread reliance on all three of these pathways for AML survival/proliferation, we propose to test the role and downstream pathways regulated by MLL1 and MLL2 in a variety of genetically defined AML animal models, as well as human leukemia lines and primary samples. Specifically, our proposal aims to 1) determine whether loss of MLL1/MLL2 or both affects AML driven by a variety of genetically-defined murine AML models, 2) identify MLL1/MLL2 regulated pathways in human AML cells along with the domains within MLL2 that contribute regulating these pathways, and 3) define the transcriptome perturbations, epigenomic states, and effect of both Mll1 and Mll2 knockout in MLL-AF9-driven AML and determine the mechanism by which they collaborate to support AML survival. These experiments will identify epigenetic vulnerabilities that may be particular to certain cytogenetic subtypes of AML or may be broadly relevant.